The intestinal mucosa maintains barrier function while allowing paracellular absorption of water and small nutrients. These opposing functions are balanced by the tight junction (TJ), which regulates paracellular permeability. The goal of this proposal is to elucidate molecular mechanisms that mediate TJ regulation in intestinal epithelium. Previous studies in isolated mammalian small intestinal mucosa and intact animals have shown that TJ permeability can be physiologically regulated by apical Na+-glucose cotransport. During the K08 award we have established a novel in vitro model of TJ regulation in the Caco-2 enterocyte-like cell line. This system was used to demonstrate that initiation of Na+-glucose cotransport causes increased TJ permeability. This led to the hypothesis that perijunctional actomyosin contraction is an intermediate in physiological TJ regulation. Evaluation of myosin II regulatory light chain (MLC) phosphorylation, a biochemical marker of actomyosin contraction, confirmed that MLC phosphorylation is necessary for this TJ regulation. The data also show that Na+-glucose cotransport activates the brush border Na+-H+ exchanger NHE3 and that inhibition of NHE3 decreases both MLC phosphorylation and TJ permeability, suggesting a common signaling pathway. Thus, the hypothesis that initiation of Na+-glucose cotransport triggers a signaling cascade that, in sequence, leads to i) NHE3 activation, ii) cytoplasmic alkalinization, iii) increased intestinal epithelial MLC kinase activity, iv) increased MLC phosphorylation, v) perijunctional actomyosin contraction, and vi) increased TJ permeability was developed. The first specific aim will focus on events that occur immediately after initiation of Na+-glucose cotransport and lead to NHE3 activation and cytoplasmic alkalinization. This will include evaluation of kinase pathways, intracellular Ca2+ and pH, and NHE3 activation. The second specific aim will concentrate on characterization of MLC kinase and its regulation. Finally, the third specific aim will use a regulated expression system to trigger MLC phosphorylation independent of proximal events, thereby allowing evaluation of structural and functional cytoskeletal and TJ modifications that follow MLC phosphorylation. These studies will provide mechanistic insight into signaling pathways that follow Na+-nutrient cotransport and cause increased paracellular permeability. These studies may also provide detailed understanding of mechanisms of altered intestinal permeability in a wide range of human diseases, from Crohn's disease to infectious enteritis, and provide targets for novel therapeutic interventions.